In mammals, kidney development involves the specification of the metanephric mesenchyme (MM), the outgrowth of the ureteric bud (UB), and its correctly patterned branching morphogenesis to generate the ureter and the renal collecting system. It is known that GDNF/Ret signaling plays a central role in these processes in mice. However, the genes that control the specification of MM and pattern the UB and its branching morphogenesis, and the regulatory mechanism of Gdnf expression in the MM are still poorly understood. The long-term objective of this proposal is to elucidate the mechanisms by which Eya1 and Six1 control normal kidney development. Defects in these genes in humans cause Branchio-Oto-Renal (BOR) syndrome, a congenital birth defect characterized by combinations of branchial, otic and renal anomalies. To understand the developmental and molecular bases of renal defects that occur in BOR syndrome, we analyzed the mutations identified in the EYA1 and SIX1 genes from BOR patients functionally and generated Eya1 and Six1 mutant mice through gene targeting. Our results indicate that Eya1 specifies the MM and is a critical regulator for Gdnf expression in the MM. We found that Eya1 acts upstream of Six1 but both gene products interact during kidney development. Furthermore, we have shown that Six1 is a critical regulator for branching morphogenesis by mediating the expression of Gdnf, Six2 and Pax2 in the MM. We also demonstrated that the mutations identified in either the EYA1 or SIX1 gene from BOR patients affected either Eya1-Six1 interaction or Six1-DNA binding, thus providing new insights into the molecular basis of renal developmental diseases in humans. This application will continue to define the molecular and developmental mechanisms by which the Eya1 and Six1 drive normal kidney development. First, we will investigate the mechanisms by which Eya1 specifies the MM and drives normal nephrogenesis. Next, we will investigate the regulation of Eya1 activity in kidney development. Lastly, we will define the mechanism by which Six1 initiates branching morphogenesis. These studies should lead to significant improvements in our understanding of the multilayer control mechanisms by Eya1-Six1 that mediate development of a normal kidney. In addition, these results will provide important insights into the developmental and molecular pathogenesis of renal defects occurring in BOR syndrome.